Department of Physiology, University of California, San Francisco, California, USA.
Department of Biochemistry & Biophysics, University of California, San Francisco, California, USA.
J Biol Chem. 2023 Dec;299(12):105362. doi: 10.1016/j.jbc.2023.105362. Epub 2023 Oct 19.
The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) compacts the RNA genome into viral ribonucleoprotein (vRNP) complexes within virions. Assembly of vRNPs is inhibited by phosphorylation of the N protein serine/arginine (SR) region. Several SARS-CoV-2 variants of concern carry N protein mutations that reduce phosphorylation and enhance the efficiency of viral packaging. Variants of the dominant B.1.1 viral lineage also encode a truncated N protein, termed N or Δ(1-209), that mediates genome packaging despite lacking the N-terminal RNA-binding domain and SR region. Here, we use mass photometry and negative stain electron microscopy to show that purified Δ(1-209) and viral RNA assemble into vRNPs that are remarkably similar in size and shape to those formed with full-length N protein. We show that assembly of Δ(1-209) vRNPs requires the leucine-rich helix of the central disordered region and that this helix promotes N protein oligomerization. We also find that fusion of a phosphomimetic SR region to Δ(1-209) inhibits RNA binding and vRNP assembly. Our results provide new insights into the mechanisms by which RNA binding promotes N protein self-association and vRNP assembly, and how this process is modulated by phosphorylation.
严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）的核衣壳（N）蛋白将 RNA 基因组压缩成病毒核糖核蛋白（vRNP）复合物在病毒粒子内。vRNP 的组装被 N 蛋白丝氨酸/精氨酸（SR）区域的磷酸化抑制。几种引起关注的 SARS-CoV-2 变体携带 N 蛋白突变，降低磷酸化并提高病毒包装的效率。占主导地位的 B.1.1 病毒谱系的变体还编码一种截断的 N 蛋白，称为 N 或 Δ(1-209)，尽管缺乏 N 端 RNA 结合域和 SR 区域，但仍介导基因组包装。在这里，我们使用质量光度计和负染色电子显微镜显示，纯化的 Δ(1-209)和病毒 RNA 组装成 vRNP，其大小和形状与全长 N 蛋白形成的 vRNP 非常相似。我们表明，Δ(1-209) vRNP 的组装需要中央无序区的富含亮氨酸的螺旋，并且该螺旋促进 N 蛋白寡聚化。我们还发现，将磷酸模拟 SR 区域融合到 Δ(1-209)上会抑制 RNA 结合和 vRNP 组装。我们的研究结果提供了关于 RNA 结合如何促进 N 蛋白自组装和 vRNP 组装的新见解，以及该过程如何被磷酸化调节。
